Therefore, this study aimed to evaluate the effects of umbilical cord extract-derived formulae on three kinds of skin cells including fibroblasts, keratinocytes, and melanocytes.. We pre
Trang 1In vitro evaluation of the effects of human umbilical cord
extracts on human fibroblasts, keratinocytes, and melanocytes
Phuc Van Pham&Loan Thi-Tung Dang&
Uyen Thanh Dinh&Huyen Thi-Thu Truong&
Ba Ngoc Huynh&Dong Van Le&Ngoc Kim Phan
Received: 6 August 2013 / Accepted: 9 October 2013 / Editor: T Okamoto
# The Society for In Vitro Biology 2013
Abstract Skin aging is the result of internal and external
factors So-called photoaging has been identified as the major
factor in skin aging Effects of photoaging include inhibition
of fibroblast and keratinocyte proliferation as well as collagen
and fibronectin expression, while activating expression of
collagenases such as matrix metalloproteinase-1 Previous
studies have shown that extracts or products from human
placenta significantly improve skin aging and chronic wound
healing However, there are few studies of umbilical cord
extracts Therefore, this study aimed to evaluate the effects
of umbilical cord extract-derived formulae on three kinds of
skin cells including fibroblasts, keratinocytes, and
melanocytes We prepared 20 formulae from intracellular
umbilical cord extracts, extracellular umbilical cord extracts,
and umbilical cord-derived stem cell extracts, as well as five
control formulae We evaluated the effects of the 25 formulae
on fibroblast and keratinocyte proliferation, and expression of collagen I, fibronectin, and matrix metalloproteinase-1 in fibroblasts and tyrosinase in melanocytes The results showed that 7.5% formula 35 was the most effective formula for promotion of fibroblast and keratinocyte proliferation At this concentration, formula 35 also induced collagen expression and inhibited matrix metalloproteinase-1 expression at the transcriptional level However, this formula had no effect on tyrosinase expression in melanocytes These results demonstrate that umbilical cord extracts can serve as an attractive source of proteins for skincare and chronic wound healing products
Keywords Fibroblast Keratinocyte Melanocyte Skin aging Umbilical cord extract
Introduction
Skin aging is an increasing concern because of its social aspect It is also an ideal model to examine aging of the body Skin aging is the result of internal and external factors So-called photoaging has been identified as the major factor in skin aging In sunlight, the ultraviolet (UV) component causes the majority of skin aging
UV radiation activates signaling pathways via protein kinases in as little as 1 h These signaling pathways reach their maximum activation after 4 h of UV irradiation (Fisher
et al 1998; Garrington and Johnson 1999) At this time, protein kinases are activated in all signaling pathways of the epidermal layer Activated protein kinases upregulate and activate transcription factors such as activating protein-1 (AP-1; composed of Jun and Fos) Subsequently, AP-1 stimulates overexpression of matrix metalloproteinase (MMP)-1, MMP-3 (stromelysin-1), and MMP-9 (gelatinase)
P Van Pham ( *):L T <T Dang:N K Phan
Laboratory of Stem Cell Research and Application, University of
Science, Vietnam National University, Ho Chi Minh City, Vietnam
e-mail: pvphuc@hcmuns.edu.vn
L T <T Dang
e-mail: dttloan@hcmus.edu.vn
N K Phan
e-mail: pkngoc@hcmuns.edu.vn
U T Dinh:H T <T Truong:B N Huynh:D Van Le
Mekostem Bank, Mekophar Chemical Pharmaceutical Joint-Stock
Company, Ho Chi Minh, Vietnam
U T Dinh
e-mail: uyendt@mekostem.com
H T <T Truong
e-mail: huyenttt@mekostem.com
B N Huynh
e-mail: levandong@yahoo.com
D Van Le
Department of Immunology, Vietnam Military Medical University,
104 Phung Hung, Ha Dong, Ha Noi, Vietnam
DOI 10.1007/s11626-013-9706-1
Trang 2that degrade collagen (Karin et al.1997; Angel et al.2001).
AP-1 also affects collagen expression in fibroblasts
Furthermore, MMP-1 triggers collagen I and collagen III
degradation After such degradation by MMP-1, collagen
fragments are continuously cleaved by MMP-3 and MMP-9
(Sternlicht and Werb2001) Therefore, upon induction by UV
light, MMPs will degrade collagen and change the structure of
the skin If collagen repair is not present at the skin, the change
of the skin structure gradually increases over time This
process is referred to as the skin aging process
UV radiation also inhibits collagen I and collagen III
synthesis (Fisher et al.2000) There are two mechanisms that
degrade collagen In the first mechanism, UV radiation
induces AP-1 expression Through binding to the transcription
factor of protocollagen, AP-1 directly interferes with collagen
expression (Chung et al.1996; Karin et al.1997) The second
mechanism involves blocking the effects of transforming
growth factor (TGF)-β (Chung et al.1996; Massague2000)
UV irradiation for 8 h results in downregulation of TGF-β
receptor type 2 expression, which decreases the effects of
TGF-β (Quan et al 2001) Collagen degradation and
downregulation of collagen expression cause a strong
reduction of collagen in the skin This reduction gradually
changes the extracellular matrix, causing the appearance of
skin wrinkles, a major characteristic of skin ageing Therefore,
the main targets of anti-aging are inhibition of collagen
degradation or MMP expression and enhancement of new
collagen synthesis
UV light also affects keratinocytes and melanocytes in the
epidermal layer Keratinocytes are the major cell type of the
skin, which account for 90% of the epidermis The main
function of keratinocytes is formation of a barrier that protects
against pathogens (bacteria, fungi, parasites, and viruses),
heat, UV light, and water loss Once a pathogen invades the
skin, keratinocytes respond by production of inflammatory
mediators such as chemokines CXCL10 and CCL2 to attract
white blood cells to the invasion sites Keratinocytes also play
an important role in filling the gaps in the skin caused by
injury Upon injury, keratinocytes migrate from the hair
follicle to the papillary for repair, but only survive temporarily,
after which keratinocytes from the epidermis migrate for cell
replacement (Claudinot et al 2005; Ito et al 2005) In
addition, epidermal keratinocytes may contribute to the
formation of de novo hair follicles from which new skin can
form to repair skin lesions (Ito et al.2007) On the other hand,
melanocytes are pigment-producing cells, namely melanin
that constitutes the skin color Through the process of
pigmentation (melanogenesis), melanocytes produce melanin
pigment in the skin, eyes, and hair In humans, melanogenesis
occurs as two processes: basal melanogenesis and activated
melanogenesis In light skin, the degree of basal
melanogenesis is low, whereas exposure to UV-B light
induces activated melanogenesis The purpose of
melanogenesis is dermal protection from UV light that can damage DNA Melanin absorbs UV light and prevents exposure to the dermis layer (Agar and Young 2005) Melanogenesis uses the amino acid tyrosine as a raw material and tyrosinase that converts tyrosine into melanin
To date, many studies have attempted to develop a product that assists aging by skin renewal The principal of anti-skin aging is inhibition of wrinkle formation and melanogenesis, and stimulation of collagen synthesis and keratinocyte proliferation Stem cells and stem cell-rich tissue extracts are considered to contain anti-skin aging factors In fact, stem cells are capable of producing a variety of cytokines such as vascular endothelial growth factor (VEGF) and hepatic growth factor One of the sources of cells commonly used in cosmetic applications is adipose-derived stem cells (ADSCs) Recent studies have shown that ADSCs secrete cytokines that perform important functions in skin regeneration (Kim et al.2011; Song et al.2011) For example, ADSCs exhibit an antioxidant effect through production of various cytokines and also secrete extracellular matrix proteins such as collagen, fibronectin, and elastin (Song
et al.2011), the major protein in soft tissues such as the skin and breast However, ADSCs are not used in cosmetic products such as creams and lotions as well as in cosmetic dermatological procedures In fact, ADSC-based production holds some limitations such as complexity and expensiveness
So that, some stem cell-rich tissue extracts were studied that can replace ADSCs
Stem cell-rich tissue extracts such as human placental extracts have been shown to stimulate the proliferation of primary cultured keratinocytes (O’Keefe et al.1985) Similar
to ascorbic acid, human placental extracts also stimulate fibroblast proliferation (Cho et al 2008) Human placental extracts also promote melanogenesis by activation of tyrosinase expression (Mallick et al.2005; Singh et al.2005; Saha et al.2006; Sarkar et al.2006) A recent study suggests that the placenta contains a number of important active peptide antioxidants that show anti-aging effects (Togashi
et al 2002) These antioxidants protect stem cells from damage caused by radiation (Kawakatsu et al 2013) Several other studies have shown that Wharton’s jelly extracts slow the aging process of stem cells through the p53 pathway and p16INK4a/pRb (Hao et al.2013)
The human umbilical cord is a rich source of stem cells including mesenchymal stem cells, endothelial stem cells, and epithelial stem cells (Gonzalez et al.2010; Kita et al.2010; Reza et al 2011a, b; Tong et al 2011; Dorronsoro and Robbins2013) In addition to stem cells, the human umbilical cord contains essential proteins for skin regeneration such as acidic fibroblast growth factor (FGF), basic FGF, epidermal growth factor (EGF), insulin-like growth factor (IGF)-I, platelet-derived growth factor (PDGF), and TGF-β (Sobolewski et al 2005) The amounts of these factors per
Trang 3gram of tissue vary from about 40 pg (EGF and PDGF) to
about 200 ng (IGF-I) (Sobolewski et al.2005)
Therefore, we considered that umbilical cord tissue extracts
may contain growth factors that stimulate collagen synthesis,
inhibit collagen degradation in fibroblasts, promote
keratinocyte proliferation, and inhibit melanogenesis of
melanocytes Our study aimed to investigate the effects of
human umbilical cord extracts on three kinds of skin cells
including fibroblasts, keratinocytes, and melanocytes
Materials and Methods
Cell phenotyping by flow cytometry Third passage fibroblasts,
keratinocytes, and melanocytes were analyzed for marker
expression by flow cytometry CD90, CD24, and CD117 were
used to confirm the fibroblasts, keratinocytes, and
melanocytes, respectively Cells at 70–80% confluency were
detached by 0.25% trypsin/EDTA and harvested as single
cells A total of 1×106 cells were stained with
anti-CD90-PE, anti-CD24-FITC, or anti-CD117-FITC antibodies at 4°C
for 30 min Then, the cells were washed twice with sheath
fluid Finally, the samples were analyzed by a FACSCalibur
(BD Bioscience, San Jose, CA) All data were analyzed as 10,
000 events in triplicate by CellQuest Pro software (BD
Bioscience)
Isolation of stem cells from the umbilical cord Human
umbilical cord samples were obtained at Tu Du Hospital and
An Sinh Hospital with informed consents Umbilical cords
used in this study met the suggested standards of NetCord and
AsiaCord The samples were digested with collagenase to
harvest the cells Stem cell populations were positive for
CD90 (mesenchymal stem cells), CD117 (epithelial stem
cells), or CD133 (endothelial stem cells) Cells positive for
FITC-conjugated anti-CD90, anti-CD117, or anti-CD133
antibody staining were sorted and considered as total stem
cells of the umbilical cord Stem cells were sorted on a
FACSJazz (BD Bioscience)
Preparation of human umbilical cord extracts and umbilical
cord-derived total stem cell extracts Umbilical cords were
washed twice with PBS and cut into small fragments The
umbilical cord fragments were washed with 0.7% NaCl and
then protease inhibitors (Sigma-Aldrich, St Louis, MO) were
added before grinding The samples were ground by
gentleMACS Dissociator (Miltenyi, Bergisch Gladbach,
Germany) following to the manufacturer’s instruction The
ground samples were centrifuged at 3,000 rpm for 20 min at
4°C The supernatant was collected, diluted at various
concentrations, and then stored at −80°C until use This
supernatant was referred to as the extracellular extract
The cell pellets were lysed by rapid freezing in nitrogen liquid and thawing in dissection solution Then, the lysates were centrifuged to remove debris These extracts were referred to as intracellular extracts The extracts were also diluted at various concentrations and stored at −80°C until use The intracellular and extracellular extracts were mixed at various ratios to prepare the formulae to treat the fibroblasts, keratinocytes, and melanocytes
The umbilical cord-derived total stem cell extract was also prepared in the same manner
MTT assay Cells (5 × 103/well) were cultured in 96-well plates in 100μL medium Twenty microliters of MTT (5 g/ L; Sigma-Aldrich) was added to each well, followed by incubation for 4 h, and then addition of 150μL/well DMSO (Sigma-Aldrich) The plates were then agitated for 10 min or until the crystals dissolved completely Absorption values were then measured at a wavelength of 490 nm and reference wavelength of 690 nm using a DTX 880 micro-plate reader (Beckman Coulter, GmbH, Krefeld, Germany)
Real-time RT-PCR Fibroblasts were analyzed for gene expression of skin aging markers including collagen type I, fibronectin, and MMP-1 with GAPDH as the internal control
In melanocytes, we investigated tyrosinase expression Total RNA was extracted as described elsewhere (Phuc et al.2011) Real-time RT-PCR was performed with an Eppendorf gradient
S thermal Cycler (Eppendorf-AG, Hamburg, Germany) Reaction mixtures of 25μL included 10 mM Tris–HCl, pH 8.3, 50 mM KCL, 1.5 mM MgCl2, 200μM dNTPs, 0.2 μM of each primer, and 1 U Taq DNA polymerase Relative expression levels were normalized to GAPDH expression and calculated using the 2−ΔCCt method PCR primers are listed Tables1and2
Statistical analysis All experiments were performed in triplicate A value of p≤0.05 was considered to be significant Data were analyzed using Statgraphics software 7.0 (Statgraphics Graphics System, Warrenton, VA)
Results
Umbilical cord extracts and total stem cell extracts We collected 130 umbilical cords with an average weight of
18 g The concentrations of the obtained total extracellular proteins and total intracellular proteins were measured by the Bradford method The extracellular and intracellular protein concentrations were 3.9 mg/mL (6,000 mL) and 2.1 mg/mL (1,500 mL), respectively By cell sorting, we also collected 1×
108total stem cells and obtained 1 mg/mL total protein
Trang 4To evaluate the effects of umbilical cord extracts, we
prepared 25 formulae of extracellular, intracellular, and total
stem cell proteins, including 11, 12, 13, 14, and 15 for
extracellular proteins; 21, 22, 23, 24, and 25 for intracellular
proteins; 31, 32, 33, 34, and 35 for mixtures of extracellular
and intracellular proteins; 41, 42, 42, 44, and 45 for total stem
cell-derived proteins; and 51, 52, 53, 54, and 55 for controls
that supplemented the dilutions The differences of formulae
from 1 to 5 in each group of proteins were different
concentrations of added proteins in the final formula A total
of 25 formulae were used to culture the fibroblasts and
keratinocytes, and investigate their effects on fibroblast and
keratinocyte proliferation
Isolation, culture, and confirmation of fibroblasts,
keratinocytes, and melanocytes Skin dermis was cultured
in DMEM/F12 supplemented with 10% FBS At days 2–4,
some fibroblasts started to adhere and exhibit a particular
shape (Fig.1A) At days 5–14, the cells rapidly proliferated
and reached 80% confluence by day 14 (Fig 1B)
Fibroblast candidates were subcultured for expansion to
obtain a more homogenous cell population (Fig 1C) To
confirm the fibroblasts and the cell population purity, we
evaluated the percentage of CD90-positive cells There
were more than 90% CD90-positive cells in the obtained cell population These fibroblasts were used in subsequent experiments
The skin epidermal layer was cultured to obtain keratinocytes After 72 h of culture, some cells appeared with
a particular shape From day 4, keratinocytes grew rapidly, co-cloned, and reached 70% confluence by day 10 (Fig.1D–F)
In flow cytometry, more than 70% of the cells expressed the epithelial marker CD24 (Fig.1L), indicating that most cells were keratinocytes
Similar to fibroblasts and keratinocytes, to isolate melanocytes, we cultured the skin epidermal layer in suitable medium with HMGS-2 After 72 h of culture, cells with a spindle shape appeared around the epidermal layer (Fig.1G) After 10 d of culture, they co-cloned, proliferated rapidly, and reached confluence by day 15 The cells were subcultured for three passages and maintained their particular shape (Fig.1H–
I) Flow cytometry results also showed that 75% of the cells expressed CD117, a specific marker of melanocytes (Fig.1M)
Effects of the 25 formulae on fibroblast proliferation The effects of the 25 formulae on fibroblast proliferation are presented in Fig 2 In most of the formulae, fibroblast
Table 1 Twenty-five formulae of extracellular, intracellular and total stem cell proteins used in this study
1 (12.5% of stock, diluted
in 0.7% NaCl)
2 (25% of stock, diluted
in 0.7% NaCl)
3 (50% of stock, diluted
in 0.7% NaCl)
4 (75% of stock, diluted
in 0.7% NaCl)
5 (100% of stock)
3 (intracellular and extracellular protein) 31 32 33 34 35
4 (total stem cell proteins) 41 41 42 43 44
5 (control: 0.7 % NaCl solution,
supplemented with protease inhibitors)
Table 2 Primer sequences
Genes Accession no Primers (5 ′-3′) References
Collagen type I NM_000088.3 F:CGGAGGAAACTTTGCTCCCC Kim et al ( 2007 )
R:CCCTTAGCACCAGTGTCTCC Fibronectin NM_212482.1 F:TGAAGAGGGGCACATGCTGA
R:GTGGGAGTTGGGCTGACTCG Matrix metalloproteinase (MMP)-1 YP_003494989.1 F:AAAATCCTGTCCAGCCCATCG
R:TTCTGTCCCTGAACAGCCCAGT Tyrosinase NM_000372.4 F:AGCACCCCACAAATCCTAACTTAC
R:ATGGCTGTTGTACTCCTCCAATC
Reinisalo et al ( 2012 ) GAPDH NM_002046 F:GGGCTGCTTTTAACTCTGGT
R:TGGCAGGTTTTTCTAGACGG
Trang 5proliferation increased gradually from days 0 to 10 Compared
with the control, some formulae inhibited cell proliferation,
including formulae 12, 13, 15, 21, 22, 23, 24, 25, and 44 Cell
proliferation rates in formulae 11, 14, and 41 were similar to
those in the control Fibroblast proliferation in formulae 31,
32, 33, 34, 35, 42, 43, and 45 was more stimulated than that in
the control The highest proliferation rate was observed in
formula 35 In this formula, after day 4, the cells started to
proliferate strongly compared with that in the other groups In
particular, compared with the other formulae, absorbance values in the MTT assay were two–fourfold higher at days 8 and 10
Effects of supplemented concentrations of formula 35 on fibroblast proliferation Next, we investigated the effects of supplemented concentrations of formula 35 on fibroblast proliferation Four concentrations of formula 35 were evaluated, including 2.5, 5, 7.5, and 10% (Fig.3) There were
Figure 1 Isolation, culture, and confirmation of fibroblasts,
keratinocytes, and melanocyte Fibroblasts exhibited a spindle shape
(A –C) and were positive for CD90 (K) Keratinocytes exhibited a round
shape (D –F) and were positive fot CD24 Melanocytes were small, exhibited a spindle shape (G –I), and were positive for CD117 (M).
Trang 6differences in the cell proliferation rate at the various
concentrations of formula 35 The proliferation rate increased
gradually from 0 to 10% formula 35 At 2.5% formula 35 in
complete medium, fibroblasts proliferated more rapidly than
those in the control (0% formula 35), but this difference was
not significant At 5, 7.5, and 10% formula 35, fibroblast
proliferation was clearly stimulated compared with that in
the control The fibroblast proliferation rates at 7.5 and 10% were similar Therefore, 7.5% formula 35 was chosen as the optimal concentration for further experiments
Effects of formula 35 on keratinocyte proliferation Next, we evaluated the effects of formula 35 on keratinocyte proliferation MTT assays showed that the various formulae had different effects on keratinocyte proliferation In most groups, keratinocytes proliferated from days 0 to 10 In general, the extracellular protein extracts (formulae 11–15) and intracellular protein extracts (formulae 21–25) lightly stimulated keratinocyte proliferation (Fig 4) The stem cell extract also increased keratinocyte proliferation The strongest proliferation was recorded in formula 35 that contained both intracellular and extracellular protein extracts
At days 6, 8, and 10, the absorbance values of MTT assays in formula 35 were significantly different compared with those
in the control
Figure 2 Fibroblast proliferation
rate in 25 formulae of umbilical
cord extracts and total stem cell
extracts at 0, 2, 4, 6, 8, and 10 d.
Fibroblast proliferation in
response to the 25 formulae The
strongest stimulating effects were
observed in formula 35.
Figure 3 Effects of various supplemented concentrations of formula 35
on fibroblast proliferation Fibroblast proliferation was similar at 7.5 and
10% formula 35.
Trang 7Effects on skin wrinkle- and melanin-related gene
expression We evaluated the effects of 7.5% formula 35 on
the expression of wrinkle-related genes including collagen I,
fibronectin, and MMP-1 Formula 35 affected collagen I,
fibronectin, and MMP-1 expression compared with that in
the control (Fig.5) Formula 35 strongly inhibited MMP-1
expression, while significantly stimulating collagen I expression (p <0.5) However, fibronectin expression showed almost no change between the formula 35 group and control (Fig 5) We also investigated the expression of tyrosinase, which has an important role in melanin synthesis, and found that formula 35 did not affect tyrosinase expression (Fig.5)
Discussion
The umbilical cord is a rich source of stem cells There are at least four stem cell-rich components of the umbilical cord, including the cord lining membrane, Wharton’s jelly, umbilical cord vein, and umbilical cord blood The stem cells
in these tissues have been isolated, cultured, and applied to treat diseases in preclinical and clinical studies In this study,
we evaluated the effects of umbilical cord extracts and umbilical cord-derived stem cell extracts on three kinds of skin cells
In the first experiment, we collected umbilical cords to isolate the stem cells and prepare umbilical cord extracts Total stem cells from the umbilical cord were isolated based
on expression of CD90 (mesenchymal stem cells), CD133 (hematopoietic stem cells as endothelial progenitor cells), and CD117 (epithelial stem cells) CD90 is highly expressed
by most mesenchymal stem cells, especially umbilical cord-derived stem cells including those in the umbilical cord lining membrane (Gonzalez et al 2010), Wharton’s jelly (Zhang
et al 2012), and whole umbilical cord (Tong et al 2011; Salehinejad et al 2012; Zhang et al 2012) CD133 is expressed in the umbilical cord vein (Yu et al.2007), whereas CD117 is also expressed in Wharton’s jelly-derived stem cells (Jo et al.2008; Montanucci et al.2011) Total stem cells were used to prepare stem cell extracts by rapid freezing and thawing We also prepared extracellular and intracellular extracts of the whole umbilical cord for use in experiments
We isolated three skin cell types including fibroblasts, keratinocytes, and melanocytes Fibroblasts were easily isolated by culturing in DMEM/F12 supplemented with 10% FBS They exhibited a particular shape and were positive for CD90 CD90, also known as Thy-1, is a cell adhesion molecule with a molecular weight of 25–35 kDa, which is expressed on certain kinds of stem cells and fibroblasts Based
on CD90 expression, Kisselbach et al (2009) removed contaminating fibroblasts (Kisselbach et al.2009) We also isolated keratinocytes and melanocytes from primary cultures
of skin tissue These cells strongly expressed CD24 (keratinocytes) or CD117 (melanocytes) and exhibited particular shapes CD24 is considered as a marker of human skin keratinocytes (Redondo et al.1998) CD117, also known
as c-kit, is an important marker of human melanocytes (Grichnik et al.1996; Norris et al.1996; Welker et al.2000)
Figure 4 Effects of the 25 formulae at 7.5% on keratinocyte proliferation
at 0, 2, 4, 6, 8, and 10 d Keratinocyte proliferation showed differences in
response to the 25 formulae The strongest stimulation of proliferation
was observed in formula 35.
Figure 5 Effects of formula 35 on skin aging-related gene expression in
fibroblasts and melanocytes (A) Formula 35 strongly reduced MMP-1
expression, while there was no significant effect on collagen I and
fibronectin expression (B ) Gene expression levels of tyrosinase and
GAPDH in the two groups Col I, collagen I; Fibro, fibronectin;
MMP-1, matrix metalloproteinase I.
Trang 8After obtaining the three kinds of skin cells, we evaluated
the effects of the 25 formulae on fibroblast and keratinocyte
proliferation, as well as expression of aging-related genes in
fibroblasts and a melanin synthesis-related gene (tyrosinase)
in melanocytes The results showed that the 25 formulae had
different effects on fibroblast and keratinocyte proliferation
However, in both fibroblasts and keratinocytes, extracellular
extracts (formulae 11–15) showed a higher stimulatory effect
on their proliferation than that of intracellular extracts
(formulae 21–25) at certain concentrations These results
demonstrated that extracellular extracts contain factors that
stimulate fibroblast and keratinocyte proliferation In fact,
some growth factors have been detected in Wharton’s jelly,
such as acidic FGF, basic FGF, EGF, IGF-I, PDGF, and
TGF-β (Sobolewski et al.2005) These factors have shown
strong stimulation of both fibroblast (Xiao et al.2012) and
keratinocyte (Meyer et al 2012; Blumenberg 2013)
proliferation In a recent study, Hao et al (2013) found that
Wharton’s jelly extracts are an ideal microenvironment for
mesenchymal stem cell culture This extract preserved
mesenchymal stem cell properties by delaying senescence
(Hao et al 2013) Intracellular extracts may not contain
growth factors because they weakly simulated proliferation
of these cells
High protein concentrations can inhibit fibroblast and
keratinocyte proliferation In this study, the extracellular
protein concentration was nearly twofold higher than that of
the intracellular proteins Therefore, at the lowest
concentration of the extracellular protein extract (formula
11), fibroblast and keratinocyte showed the strongest
proliferation Moreover, these proliferation rates decreased
gradually as the concentration was increased gradually from
formulas 11 to 15 The protein concentration of the
intracellular extract was low Therefore, formula 24 showed
the strongest stimulation of both fibroblast and keratinocyte
proliferation These results indicated that high or low protein
concentrations of extracts can be unfavorable for cells because
of the osmotic strength of the medium
Mixtures of intracellular and extracellular extracts
stimulated fibroblast and keratinocyte proliferation We found
that formulae 31–35, which were mixtures of intracellular and
extracellular protein extracts, strongly enhanced cell
proliferation In particular, fibroblasts cultured with formula
35 showed very high proliferation at days 8 and 10 These
results demonstrated that combinations of intracellular and
extracellular proteins had the most significant effects on
fibroblast and keratinocyte proliferation
In the first experiment using fibroblasts, we determined that
7.5% formula 35 in complete medium had the optimal effect
on fibroblast proliferation Therefore, in the next experiment
using keratinocytes, we evaluated the effects of the 25
formulae at 7.5% in complete medium The results indicated
that 7.5% formula 35 in complete medium stimulated both
fibroblast and keratinocyte proliferation Next, we evaluated the effects of 7.5% formula 35 on the expression of aging-related genes including collagen I, fibronectin, and MMP-1 Compared with the control, formula 35 significantly stimulated collagen I expression and strongly inhibited MMP-1 expression These effects would increase collagen I production in fibroblasts that can reduce skin aging, especially wrinkles In fact, the main cause of wrinkle formation and skin aging is a decrease of collagen synthesis and increase of MMP expression MMPs are proteinases that disrupt the skin structure by collagen, laminin, fibronectin, and elastin degradation In vivo, the activity of MMP-1 is regulated by certain proteins and MMP inhibitors When MMP-1 is overexpressed, the skin structure changes and forms wrinkles (Rittie and Fisher 2002; Hornebeck 2003) Therefore, inhibition of MMP-1 expression is important for prevention
of wrinkles Formula 35 inhibited MMP-1 expression because
of the presence of TGF-β In a previous study, Alvares et al demonstrated that TGF–β induces a 25–50% decrease of MMP-1 expression (Alvares et al.1995)
Finally, we evaluated the effect of formula 35 on tyrosinase expression in melanocytes Tyrosinase is an important enzyme that converts tyrosine into melanin that darkens skin However, formula 35 had no effect on tyrosinase expression, indicating that the components of formula 35 could not lighten skin This result is in contrast to the effect of placental extracts
in a previous report by Pal et al (2002) They found that placental extracts stimulate both in vitro and in vivo melanogenesis (Pal et al 2002) The placental protein/ peptide fraction also induced melanogenesis in vitro This fraction was found to increase tyrosinase expression at transcription and translation levels in B16F10 cells (Sarkar
et al.2006)
There are a few studies of umbilical cord extracts In contrast, many studies have investigated the effects of placental extracts on skin cells Similar to our data, placental extracts also induce fibroblast proliferation (Cho et al.2008) This effect is similar to that of ascorbic acid, but placental extracts do not significantly increase TGF-β1 protein expression (Cho et al 2008) Therefore, placental extracts have a beneficial role in chronic non-healing wounds (Shukla et al.2004; Tiwary et al.2006) This beneficial effect
is related to an increase of TGF-β in the early phase of wound healing and VEGF in the late phase (Hong et al.2010) This wound healing activity may be induced by the biological activity of NADPH (Datta and Bhattacharyya 2004) and fibronectin type III-like peptide (Nath and Bhattacharyya
2007) that are present in placental extracts In a recent study,
an aqueous extract of human placenta exhibited strong gelatinase/collagenase activity in zymography, which had thermostable intrinsic collagenase activity and wide physiological relevance in degrading and remodeling collagen Therefore, placental extracts may be used for wound
Trang 9healing and the treatment of pelvic inflammatory diseases (De
et al 2013) In addition, placental extracts regulate the
immunological response in allergic skin disease (Kim et al
2010), reduce inflammation by strong gelatinase/collagenase
activity (De et al 2013), and significantly inhibit the
production of nitric oxide, tumor necrosis factor-α, and
cyclooxygenase-2 (Lee et al.2011)
Conclusion
Umbilical cords are considered as medical waste However,
they contain a rich source of stem cells and strong biologically
active factors This study showed that umbilical cord extracts
of extracellular and intracellular proteins in formula 35 had
some beneficial effects on two kinds of skin cells, fibroblasts,
and keratinocytes At 7.5% formula 35, the umbilical cord
extracts efficiently stimulated fibroblast and keratinocyte
proliferation At this concentration, formula 35 also stimulated
collagen I expression and inhibited MMP-1 expression in
fibroblasts However, formula 35 did not affect tyrosinase
expression in melanocytes These results indicate that the
umbilical cord extracts in formula 35 can be used in skincare
and wound healing products
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